Nephrol Dial Transplant (2006) 21: 1803–1808
doi:10.1093/ndt/gfl066
Advance Access publication 23 March 2006
Original Article
Primary organ function of warm ischaemically damaged porcine
kidneys after retrograde oxygen persufflation*
Jürgen W. Treckmann1, Andreas Paul1, Stefano Saad2, Julia Hoffmann3, Karl-Heinz Waldmann3,
Christoph E. Broelsch1 and Manfred Nagelschmidt4
1
Department for General, Visceral and Transplantation Surgery, University Hospital of Essen, Hufelandstr. 55, 45122 Essen,
Clinic for Visceral, Vascular and Transplantation Surgery, University of Witten Herdecke, Hospital Cologne Merheim,
Ostmerheimer Str. 200, 51109 Cologne, 3Clinic for Swine, Small Ruminants, Forensic Medicine and Ambulatory
Services, Veterinary School of Hannover, Bischofsholer Damm 15, 31073 Hannover and 4Institute of Experimental
Medicine, University of Cologne, Robert-Koch-Str. 10, 50931 Cologne, Germany
2
Abstract
Background. As warm ischaemic damage is a major
reason for the loss of donor organs, an experimental
study was performed in order to evaluate retrograde
oxygen persufflation (ROP) as a method to extend the
warm ischaemic tolerance of kidneys.
Methods. Kidneys of 32 pigs were exposed to warm
ischaemia for 60, 90 or 120 min. Then, 16 kidneys
were subjected to ROP for 4 h at 4 C and 16 controls
were stored in cold UW-solution, followed by autotransplantation.
Results. Only in the group with 60 min warm ischaemic time and ROP did all animals survive the observation period of 7 days. In all other groups some animals
died due to anuria. Short-term survivors in these
groups had significantly higher creatinine levels.
Conclusions. In this setting, ROP was superior to cold
storage when applied after 60 min of warm ischaemia.
Clinical evaluation of ROP in the setting of marginal
donors and non-heartbeating donation is recommended.
Keywords: kidney transplantation; non-heartbeating
donors; retrograde oxygen persufflation;
warm ischaemia
Introduction
There is still a major discrepancy between cadaveric
organs available for transplantation and the actual
demand [1] resulting in increasing waiting times for
Correspondence and offprint requests to: Jürgen Treckmann,
MD, Klinik für Allgemein- und Transplantationschirurgie,
Universitätsklinikum Essen, Hufelandstr. 55, 45122 Essen,
Germany. Email: [email protected]
renal transplantation by an average of 3 years in
patients on dialysis in Germany and other countries.
Attempts to increase the number of living donor
transplantations and the use of marginal donor organs
are currently the most effective strategies in order
to improve this intolerable condition. Other strategies
include utilization of organs from non-heartbeating
donors (NHBD) [2]. The NHBD programmes have
already been successfully established in several countries (e.g. USA, GB, Spain, The Netherlands) while
still restricted by Federal Laws in other countries
(e.g. Germany).
Preservation itself has the potential to sustain or even
improve organ function. Currently, preservation is
done by simple cold storage (CS) and in the setting
of NHBD-programmes by machine perfusion (MP),
at best performed as hypothermic pulsatile perfusion
[2–4]. Our previous experiments have shown that
preservation with venous systemic oxygen persufflation
in combination with anti-oxidative treatment can
restore viability of warm ischaemically damaged livers
in an in-vitro model of isolated rat liver perfusion [5–8].
We also observed a restitution of organ function
of transplanted porcine livers after 60 min of warm
ischaemia, when retrograde oxygen persufflation
(ROP) had been applied [9].
Already in the 1980s, ROP was performed to
preserve kidneys in experimental studies, but because
of the development of modern perfusion solutions,
additional treatment during preservation as MP or
ROP seemed to be unnecessary. Under these conditions
the clinical approach of Rolles et al. [17] fell into
oblivion, who at first reported a successful application
of ROP in kidney transplantation. The increasing use
of organs of marginal donors and the establishment of
NHBD programmes prompted the prosecution of
experimental work to ameliorate the quality of organs
ß The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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1804
J. W. Treckmann et al.
with ROP, which was optimized by the addition of the
scavenger superoxid dismutase (SOD) as a result of
experimental studies in animals [10,11].
Our successful studies in porcine livers encouraged
us to evaluate ROP systematically in a model of experimental kidney transplantation, also because clinical
implementation of this method should be possible in
this setting than in liver transplantation.
So we performed a controlled study in a porcine
model of autotransplantation followed by contralateral
nephrectomy in order to compare standard treatment
(CS) with the effect of ROP on primary function of
warm ischaemically damaged kidneys.
Subjects and methods
Study design
Thirty-four female German landrace pigs with a mean body
weight of 27 kg were used for the trial. The maximal warm
ischaemic time (WIT), which allowed restitution of primary
renal function by oxygen persufflation was evaluated creating
six experimental groups. The WIT was 60 min in the groups 1
and 2, 90 min in the groups 3 and 4 and 120 min in the
groups 5 and 6. Oxygen persufflation was administered to the
groups 1, 3 and 5. The experimental groups 2, 4 and 6 received
only CS without additional oxygen and were used as control
(Table 1). The experiment was started with the shortest WIT,
thus allowing to stop the study when the WIT became too
extended to restore organ function.
Therefore, randomization of the animals was only performed between the groups with identical WIT. Usually two
different teams performed explantation and implantation
of the kidneys. In these cases, the second team was blinded
with respect to the type of preservation of the organs.
Operation and treatment of the kidneys
Under general anaesthesia with ketamine, fentanyl,
pancuronium bromide, oxygen and nitrous oxide, the blood
vessels and the ureter of the left kidney were clamped and
the organ was subjected to complete in-situ warm ischaemia
with the abdomen covered. Then the kidney was removed and
flushed anterograde with cold Ringer’s solution containing
10.000 IU heparin followed by UW-solution (ViaspanÕ ). In
the ROP groups, 12.500 IU of superoxid dismutase (SOD,
Sigma Chemicals, St Louis, MO) were infused with the last
20 ml of the UW-solution. All explanted organs were stored
Table 1. Survival in the different treatment groups
Group
Treatment
n
7 day survival n (%)
1
2
3
4
5
6
60 min WIT, ROP
60 min WIT, CS
90 min WIT, ROP
90 min WIT, CS
120 min WIT, ROP
120 min WIT, CS
6
7
7
6
3
3
6
4
5
5
1
1
(100)
(57.1)
(71.4)
(83.3)
(33.3)
(33.3)
ROP, retrograde oxygen persufflation; CS, cold storage.
for 4 h in UW solution at 4 C. During this time, the ROP
to the kidneys was administered as described [5].
Briefly, filtered and humidified pure gaseous oxygen was
given through the renal vein with a pressure of 18 mmHg.
About 15–20 small pinpricks with a depth of about 1 cm were
set with a fine acupuncture needle into the surface of the
organ to allow the gas to leave the microvasculature. When
the oxygen flow was installed, it was checked whether there
was persufflation of all areas. Otherwise additional pinpricks
were added. The control kidneys were stored in cold UWsolution without further treatment with the exception that
they also received pinpricks in order to mask their treatment.
After 4 h of treatment, the explanted kidneys were flushed
anterograde with cold Ringer’s solution and autotransplanted
into the pigs which were still under anaesthesia. The right
kidney was removed leaving the clamped vessels as long as
possible. Then the left kidney was implanted contralaterally
constructing an end to end anastomosis of the vein and of
the artery (7/0 Prolene). After removal of the clamps and
correction for possible leakages, the ureter was connected
(5/0 PDS) and checked for free passage with the aid of a
canula. Finally, the kidney was placed into the retroperitoneal
pocket which was closed with four sutures (3/0 Vicryl).
A suprapubic catheter was inserted into the bladder and
fixed, before the abdomen was closed. The catheters of the
vena jugularis interna and the arteria carotis communis were
left in place and protected by an adhesive tape. At the end of
the operation, the animals received a subcutaneous injection
of 50 mg tramadol and an intravenous injection of 40 mg
furosemide. Post-operatively 500 mg ampicillin and 50 mg
tramadol were administered daily. In most cases, the animals
were able to drink on the day of the operation and eat on
the first post-operative day. If not, they received infusions
of 20% glucose and 0.9% NaCl.
Post-operative management and data collection
Animals without primary renal function were sacrificed, if
they developed a very bad general status (unable to move,
apathic, plasma potassium >9 mmol/l). Animals with postoperative complications, which were not specifically related
to the transplantation such as sepsis and ileus, were excluded.
Daily blood samples were taken from the arterial and
venous catheters and—if possible—samples of urine were
collected. At the end of the observation period (maximally
7 days) the animals were anaesthetized and a relaparotomy
was performed in order to examine the kidney in situ. The
kidney was adjudged concerning colour, perfusion defects
and arterial or venous thrombosis. The anastomotic sites
of both vessels were excised and inspected for technical
problems. Then the organ was removed and the animal
sacrificed for autopsy.
Tissue samples of the kidney were freeze-dried for
determination of lipid peroxidation according to Ohkawa
et al. [12] or transferred into formalin for histological
examination (PAS-staining, according to Leonhardt [13]).
Histological criteria of kidney damage were hydropic swelling
of epithelium, loss of brush border, denatured basal
membrane, tubular effusions, dilated tubuli, reduced epithelium, necrosis of single cells with loss of nucleus.
The main endpoint of the study was the primary function
of the transplanted kidneys, which was characterized by urine
production and survival and quantitated by the determination
Retrograde oxygen persufflation in porcine kidneys
1805
The data presented are expressed as mean±SD. Differences
between the groups were detected with one way ANOVA and
post hoc least significant difference (LSD) tests. For evaluation of the primary endpoints, the maximum levels during the
observation period and the final values obtained immediately
before the death of the animals were used. P 0.05 was
considered significant.
Results
16
14
12
10
8
6
4
b
2
0
60 ROP
90 ROP
90 CS
120 ROP
120 CS
250
200
150
c
100
50
0
60 ROP
60 CS
90 ROP
90 CS
120 ROP 120 CS
Fig. 3. Plasma urea maximum levels (mean±SD). cSignificantly
different from group 90 CS, 120 CS.
25
250
20
200
15
150
10
60 CS
Fig. 2. Plasma creatinine at the end of trial (day 7 or death of
animal) (mean±SD). bSignificantly different from group 60 CS,
90 ROP, 120 ROP, 120 CS.
mg/dl
mg/dl
Two animals had to be excluded from the study. One with
60 min WIT and ROP and one with 90 min WIT and CS
developed an ileus and had to be sacrificed on post-operative
days 4 and 6.
Following warm ischaemia, anterograde flushing of the
kidneys was easy in all cases. No clots could be observed. No
bleeding occurred from the pinpricks on the kidney surface.
In the group with 60 min WIT and ROP (group 1), all
six animals survived, whereas in all other groups some
animals were lost due to uraemia (Table 1).
The animals of group 1 produced urine over the complete
observation period and their maximum creatinine levels in
plasma were significantly lower than in the other groups
(Figure 1). They all reached normal creatinine levels with a
very small variance after 7 days. Concerning the final values,
group 1 showed a significant advantage when compared with
the groups 2, 3, 5 and 6 (Figure 2). However, even in the
groups with 90 min WIT and ROP (group 3) and with 90 min
WIT and CS (group 4), there were 2/5 and 3/5 animals
with nearly normal plasma creatinine. After 120 min WIT
and ROP (group 5) or CS (group 6), there was one animal
out of three in each group with highly elevated plasma
creatinine, but with urine production and already slightly
decreasing creatinine levels at the end of the observation
period.
Comparing only the animals which produced urine
supported the aforementioned findings. In spite of the
reduced number of the surviving pigs, the maximum
creatinine levels were significantly lower in group 1 when
compared with the groups 3 and 6.
mg/dl
Statistics
Determination of urea and potassium in plasma of all
animals resulted in patterns similar to creatinine. In group 1,
the maximum urea values were significantly lower than in
groups 4 and 6 (Figure 3), and the maximum values of
potassium showed a significant lower level when compared
with the groups 2, 3, 5 and 6 (data not shown). Evaluation
of the final values of urea in plasma showed a significant
advantage of group 1 compared with group 2–6 (Figure 4).
Evaluation of the final values of potassium did not result
in significant differences.
All the animals with urine production exhibited some
degree of proteinuria. When comparing the maximum levels,
group 1 showed no difference to the groups 2 through 6.
At the end of the trial only one pig still had protein >2 g/l
in its urine (90 min WIT, CS: 19 g/l). Even the two pigs
surviving in groups 5 and 6 no longer had proteinuria
(Figure 5).
As high concentrations of oxygen may damage cellular
membranes by oxygen-free radicals, lipid peroxidation was
mg/dl
of plasma creatinine (Merckotest Creatinin, Merck, Darmstadt,
Germany), urea (Merckotest Harnstoff, Merck, Darmstadt,
Germany), potassium (Blood Gas Analyzer, Gem Premier
5300, Mallinckrodt, Hennef, Germany) and protein in urine
(BIO-RAD Protein Assay, BIO-RAD Laboratories, Munich,
Germany). Additionally, a blood cell count was done.
100
a
50
5
d
0
0
60 ROP
60 CS
90 ROP
90 CS
120 ROP 120 CS
Fig. 1. Plasma
creatinine
maximum
levels
a
Significantly different from all other groups.
(mean±SD).
60 ROP
60 CS
90 ROP
90 CS
120 ROP 120 CS
Fig. 4. Plasma urea at the end of trial (day 7 or death of animal)
(mean±SD). dSignificantly different from all other groups.
1806
J. W. Treckmann et al.
measured in kidney samples of the surviving animals and in
some of the removed right kidneys as a control. The results
are presented in Table 2.
Up to 90 min WIT there were no differences between fresh
untreated kidneys and the treated kidneys whether cold
stored or persufflated. According to these data, treatment
with oxygen did not induce increased lipid peroxidation
in these groups. Both animals surviving 120 min WIT of the
kidney showed increased levels of malondialdehyde, with the
persufflated kidney far exceeding the level of the cold stored
kidney.
Judged by the macroscopic aspect, the urine producing
kidneys did not differ significantly. They all showed a reddishbrown colour and a soft-elastic consistency. The kidneys of
the anuric animals (n ¼ 10) appeared less or more necrotic
depending on the time elapsed since transplantation. In
these animals, we found four kidneys with arterial or venous
thrombosis and one with clots in both vessels. Concerning the
anastomoses no technical problems could be observed.
The remaining five necrotic kidneys were completely free
of thrombotic vessel obstruction.
Histologically, the urine producing kidneys exhibited
only minimal signs of tissue damage. Group 1 seemed
nearly completely regenerated within the observation period
however, in general group specific differences could not be
ascertained.
Discussion
The method of retrograde oxygen persufflation,
primarily discovered because of an experimental
g/l
9
8
7
6
5
4
3
2
1
0
60 ROP
60 CS
90ROP
90 CS
120 ROP
120 CS
Fig. 5. Proteine in urine at end of trial (day 7 or death of animal)
(mean±SD).
Table 2. Lipid peroxidation of kidney tissue determined on day 7
after transplantation (nmol malondialdehyde/g dry weight)
Group
Treatment
n
nmol malon-dialdehyde/g
Mean±SD
1
2
3
4
5
6
7
60 min WIT, ROP
60 min WIT, CS
90 min WIT, ROP
90 min WIT, CS
120 min WIT, ROP
120 min WIT, CS
fresh kidney
6
4
5
4*
1
1
5
99.9±62.7
65.5±23.8
85.4±62.4
75.4±11.5
424.2
170.1
79.5±30.0
ROP, retrograde oxygen persufflation; CS, cold storage.
*one sample was lost.
mistake, was utilized successfully in different
approaches in animal experiments to restore organ
function of heart, kidney and liver [11]. It has been
shown that the energy status of the organs can be
maintained under persufflation and that the treated
organs can restore their function following transplantation [11]. Ventilation of warm ischaemic damaged lungs
led to comparable good results [14].
In this study, the effects of ROP on the restitution
of the kidney in a large animal experiment in different
WITs were examined.
In our experimental setting, ROP in combination
with SOD was beneficial for functional restitution of
warm ischaemically damaged kidneys. There was a
significant positive effect after 60 min of warm ischaemia when compared with the appropriate groups with
CS. However, with the extension of the WIT to 90 and
120 min, we could find no advantage of our ROP
method.
For human NHBD, the maximum acceptable WIT
is considered to be less than 30–40 min [2]. In our set
of experiments, ischaemic tolerance was surprisingly
much higher than expected. After 90 min of warm
ischaemia without in situ anticoagulation, 10 of
13 kidneys had moderate or good initial function
regardless of the conservation method applied. Even
after 120 min of WIT, 2/6 kidneys functioned primarily.
Nevertheless, we had to stop further evaluation in
these groups because of missing approval of the animal
rights committee, since it was not expected that ROP
had a relevant positive effect after 120 min of warm
ischaemia.
There are several explanations for the extended
ischaemic tolerance we observed, at least in some of
the kidneys. Ischaemic tolerance of porcine kidneys
per se might be higher or acute. Ischaemic tolerance of
porcine kidneys per se might be higher or acute complete ischemia has different effects when compared with
the situation of NHBD with a preceding depression of
circulation including activation of a cascade of cytokine
release. Another explanation could be that the effects of
warm ischaemia injury of the kidney so far described
are simply overestimated.
Consistent with our results, canine kidneys have been
transplanted successfully by others after 60 min WIT
and 24 h of ROP [15], and in an experimental study of
Leone et al. [16], a maximum time of 50 min of WIT was
described for porcine kidneys in an NHBD situation
without additional treatment. Rolles et al. [17] transplanted kidneys in 1984 in 10 patients with a maximum
WIT of 55 min and a median time of ROP of 21.5 h.
Although several experimental studies [18,19] and the
clinical pilot trial of Rolles et al. [17] have demonstrated
beneficial effects of ROP, this method has not yet
reached the status of routine clinical application.
An additional advantage of our ROP treatment
consists in the administration of SOD as a scavenger
for radicals. It is known that reperfusion or reoxygenation after a period of ischaemia affects the vascular
system by generation of oxygen free radicals. This can
be counteracted by treatment with oxygen radical
Retrograde oxygen persufflation in porcine kidneys
scavengers such as SOD or allopurinol. In a study on
rat livers, our group has shown that addition of SOD
before ROP reduces reperfusion injuries as indicated
by decreased lipid peroxidation without affecting the
positive effects of oxygen persufflation on the energy
status [10,20]. For this reason, we have integrated these
two principles of organ protection in our concept of
ROP. The groups which underwent CS did not receive
SOD because we aimed to compare our method
with the standard procedures currently used in organ
preservation.
In this trial, the animals in the groups 1–4 showed
normal levels of lipid peroxidation, meaning that in
these groups gaseous oxygen did not cause damage.
The results of the animals in group 5 and 6 raise the
impression that SOD may not be able to prevent
oxidative damages when the WIT is extended beyond
90 min, but there was just one animal in each group.
Concerning these results, it has to be considered
that malondialdehyde was measured on day 7 after
transplantation and therefore, no conclusions on the
status directly after transplantation can be drawn.
Answers to this question require examination of
kidneys directly after preservation.
The cold ischaemic time (CIT) of 4–5 h with 4 h
treatment as applied in this study is rarely achieved
in clinical practice with a mean CIT of about 18 h for
kidneys of deceased donors in Germany. So, it has to
be questioned whether the beneficial effects of ROP
demonstrated in this study might be transferred to the
average clinical situation. Additional studies with
longer CIT should be performed. For canine kidneys,
and rat livers, it has already been shown that
treatment with ROP has comparable beneficial effects
whether applied for 2 h or for 24 h [11]. So clinical
approaches with short-term ROP followed by a long
cold preservation seem possible.
Concerning the groups with 60 min WIT, our
results agree with previously published reports
[5–9,11,15,17–19,21,22], demonstrating that ROP is
an appropriate method to optimize kidney function
in cases where delayed or no function is expected.
In addition to the good functional parameters, the
histological features of group 1 point to an accelerated
regeneration after WIT possibly as a result of a better
energy status. However, extension of the WIT to 90 and
120 min could not be successfully compensated for
by this method. Even worse, group 4 with 90 min WIT
followed by CS without ROP is almost as effective
as the 60 min WIT followed by CS with ROP, where
five out of six animals survived. Nevertheless, the
maximum level of plasma creatinine and the level of
plasma creatinine at the end of the trial in group 4 were
significantly higher than in group 1 with 60 min WIT
and ROP. From these findings, we have to conclude
that for the pig kidney, ROP is only beneficial after
the short WIT. As outlined earlier, porcine kidneys
might have a higher ischaemic tolerance than human
kidneys, which might be responsible for the fact that
the advantage of ROP could only partially be assured
after 90 min WIT.
1807
Thus we have to draw further conclusions carefully.
In conclusion of our present results and those
obtained from the liver model [9], we suggest that
ROP is superior to CS at least for the limited WIT.
Organ quality is significantly improved as shown by
the functional parameters. Therefore, ROP seems
an appropriate method to be used in cases of kidneys
which are expected to develop delayed graft function.
Whether ROP might be able to overcome longer WIT
has to be studied with human kidneys preferentialy in
comparison with MP. If ROP is also benefical for the
human kidneys, then it would represent a simple,
low-cost measure for organ restitution after severe
warm ischaemic damage and perhaps other forms
of damage, which might extend our possibilities to
recruit organs of extended criteria donors or NHBD
for transplantation [2,4].
Acknowledgements. The authors gratefully acknowledge the
valuable practical help of Simone Hess and Monika Strünker
during the perioperative setting. The work is supported by the
Deutsche Forschungsgemeinschaft (grant no. Pa 776/1-1) and by
the Köln Fortune Program (Faculty of Medicine, University of
Cologne, Germany).
*Results have been in part presented at the annual ATS meeting,
3 June 2003, Washington DC and at the Surgical Forum of the
annual meeting of the German Society of Surgery, 30 April 2003.
Conflict of interest statement. None declared.
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Received for publication: 11.6.05
Accepted in revised form: 3.2.06